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Infection and Immunity, May 2005, p. 2628-2643, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2628-2643.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Microarray and Proteomics Analyses of Human Intestinal Epithelial Cells Treated with the Aeromonas hydrophila Cytotoxic Enterotoxin

C. L. Galindo, A. A. Fadl, Jian Sha, L. Pillai, C. Gutierrez Jr., and A. K. Chopra*

Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1070

Received 13 October 2004/ Returned for modification 6 December 2004/ Accepted 21 December 2004

We performed microarray analyses on RNA from human intestinal epithelial (HT-29) cells treated with the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to examine global cellular transcriptional responses. Based on three independent experiments, Act upregulated the expression of 34 genes involved in cell growth, adhesion, signaling, immune responses (including interleukin-8 [IL-8] production), and apoptosis. We verified the upregulation of 14 genes by real-time reverse transcriptase-PCR and confirmed Act-induced production of IL-8 by enzyme-linked immunosorbent assay on supernatants from nonpolarized and polarized HT-29 cells. Maximal production of IL-8 in response to Act required the presence of intracellular calcium, since chelation of calcium with BAPTA-AM significantly reduced Act-induced IL-8 production in HT-29 cells. We also examined activation of mitogen-activated protein kinases and, as demonstrated by Western blot analysis of apical side-treated polarized HT-29 cells, Act induced phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 1/2. In addition, KinetWorks proteomics screening of whole-cell lysates revealed Act-induced phosphorylation of cyclic AMP-response element binding protein (CREB), c-Jun, adducin, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) and decreased phosphorylation of protein kinase B{alpha}, v-raf-1 murine leukemia viral oncogene homolog 1 (i.e., Raf1), and STAT1. We verified activation of CREB and activator protein 1 in polarized cells by gel shift assay. This is the first description of human intestinal epithelial cell transcriptional alterations, phosphorylation or activation of signaling molecules, cytokine production, and calcium mobilization in response to this toxin.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical Research Building, 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-1070. Phone: (409) 747-0578. Fax: (409) 747-6869. E-mail: achopra{at}utmb.edu.

Editor: J. T. Barbieri

Infection and Immunity, May 2005, p. 2628-2643, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2628-2643.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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